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Page 1
Tips and Tricks of Faster LC Analysis Without Capital InvestmentIt may be easier than you think!
Agilent Technologies
Page 2
There Are Many Reasons to Reduce Analysis Time
•Run More Samples/Day
•Reduce Mobile Phase Use
•Reduce Mobile Phase Waste Disposal
•Better Utilize Existing Resources in Instruments and Personnel
•Implement “HB5”* Work Schedule
(* “HB5” Home By 5, Ray Lombardy, Agilent Technologies, Inc. 2007)
At What Cost and Difficulty?
That Depends on What You Want to Accomplish!
2
Page 3
How Fast Do You Want To Go?Your Answer Will Determine the Cost of Increased Speed
•2 to 3 X increase in Speed – Easy
•5-10 X Increase – May Require Instrument Tweak
•10 X + Increase – Will Require Instrument Upgrade and Tweak
What Are the Paths to LC Method Speed Gains?
•Most Speed From Column and Particle Size Optimization
•Instrument Optimization Will Enable or Boost Those Gains
The Following Discussion is Designed to
Simplify the Process - Often at No Cost!
Page 4
How Do Small Particles Help Speed Up Methods?
•Increase Kinetics of the separation
•Increase Efficiency (N) in Same Length Column
•Allow Shorter Column to Match Efficiency of Longer Column
• Provide Expanded Flow Rate Range Without Loss of Efficiency
3
Page 5
Trick: Use Smaller Particles For More Efficient Separations Across a Wide Flow Rate Range
Small Particle Columns including Monolith
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Dimensions: 4.6 x 50/30/20mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0µL Octanophenone in Eluent
Volumetric Flow Rate (mL/min)
HE
TP
(cm
/pla
te)
ZORBAX 5.0µmZORBAX 3.5µmVendor A 2.5 µmVendor B 2.0 µmZORBAX 1.8µmMonolith
Page 6
ISOCRATIC ELUTION
4
Page 7
Tip: Short Column/Smaller Particle Maintains Rs Trick: Tailor Column Dimensions and Particle Size
to Analysis NeedsColumnLength(mm)
Resolving Power
N(5 µm)
Resolving Power
N(3.5 µm)
ResolvingPower
N(1.8 µm)
Typical Pressure
Bar (1.8 µm)
150 12,500 21,000 32,500 >400
100 8,500 14,000 24,000 >400
75 6000 10,500 17,000 320
50 4,200 7,000 12,000 210
30 N.A. 4,200 6,500 126
15 N.A. 2,100 2,500 55
AnalysisTime
PeakVolume
Analysis Time*
-33%
-50%
-67%
-80%
-90%SolventUsage
* Reduction in analysis time compared to 150 mm column• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID
Page 8
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Time (min)
5
4
32
1
0 1 2 3 4 5 6Time (min)
5
432
1
Goal: Run Current Method 2X FasterTrick: ½ Column Length and Smaller Particle = ½ Tim e
Mobile Phase: 45% 25 mM NaH2PO4, pH 3.0
55% MeOHFlow Rate: 2.0 mL/min.Temperature:35°CDetection: UV 254 nmSample: Cardiac Drugs
1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine
Rapid ResolutionStableBond SB-C184.6 x 75 mm, 3.5 µm
StableBond SB-C184.6 x 150 mm, 5 µm
Analysis Time:5.2 minAnalysis Time:
11.7 min
5
Page 9
Goal: Run Current Method 3X Faster Trick: 1/3 Column Length and Smaller Particle = 1/3 Time
Original methodZORBAX LC column Extend-C184.6 x 150 mm, 5 µm3 µL inj.
min0 2 4 6 8 10
mAU
0
50
100
150
200
250
min0 2 4 6 8 10
mAU
-20
0
20
40
60
80
100
120
Mobile phase: (70:30) MeOH: 50 mM pyrrolidine buffer Flow = 1.0 mL/min, Temp. : ambient
ZORBAX RRHT column Extend C184.6 x 50 mm, 1.8 µm1 µL inj.
12
2 3
5
4
4
5
3
16
6
3x faster
Page 10
How Long Can the Column Be with MeOH?System Pressure With MeOH/Water
Percentage of MeOH (%)
Pressure (bar)
0 142
10 169
20 203
30 229
40 249
50 252
60 240
70 216
80 184
90 145
100 98
4.6 x 50mm Column with 1.8um particles at 1 ml/min, 30oC
Pr essur e cur ve
050
100150200250
0 20 40 60 80 100per cent age of MeOH
pres
sure
•Use P of 4.6 x 50mm as baseline
•Multiply P by L col2 / Lcol1
•If P is Lower than Max P of Instrument you can run that Length
6
Page 11
How Long Can the Column Be with ACN?System Pressure with ACN/Water
Percentage of ACN (%)
Pressure (bar)
0 142
10 154
20 159
30 156
40 150
50 135
60 120
70 104
80 88
90 70
100 59
4.6 x 50mm Column with 1.8um particles at 1 ml/min, 30oC
Syst em pr essur e wi t h t he change ofper cent age of ACN
0
50100
150
0 50 100Per cent age of ACN
Pres
sure
•Use P of 4.6 x 50mm as baseline
•Multiply P by Lcol2 / Lcol1
•If P is Lower than Max P of Instrument you can run that Length
Page 12
More Resolution in Original Method than NeededTip: 1.8u, Very Short Column, Faster Flow 5-10X Fas ter Isocratic on Instrument with 400bar Pressure Limit
.
4.6 x 250 mm, 5 µm
min0 5 10 15 20 25 30
4.6 x 100 mm, 3.5 µm
4.6 x 30 mm, 1.8 µm 1 mL/min
4.6 x 30 mm, 1.8 µm2 mL/min
min0 5 10 15 20 25 30
min0 5 10 15 20 25 30
min0 5 10 15 20 25 30
0.5 1 1.5 2 2.5
29.65
12.71
4.15
2.09
1 23
1
1
1
2
2
2
3
3
3
4
4
4
4
Rs(1,2) = 4.8
Rs(1,2) = 3.5
Rs(1,2) = 3.3
Rs(1,2) = 3.1
Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RTDetection: UV 230 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone
N=22680
N=11691
N=6104
N=6568
N=6460
N=6463
N=21848
7
Page 13
Tip: Constant Linear Velocity Maintains EfficiencyTrick: Reduce Flow Rate When Reducing Column Diamet er
1
min0 5 10 15 20 25 30 35
mAU
0
25
50
75
100
125
150
175
min0 5 10 15 20 25 30 35
mAU
0
25
50
75
100
125
150
175
Mobile Phase: 25% methanol in 0.4% Formic Acid
ZORBAX SB-C18, 4.6 x 250 mm, 5 µm, 1 mL/minSolvent Used: 34 mL
ZORBAX SB-C18, 3.0 x 100 mm, 3.5 µm, 0.425 mL/minSolvent Used: 5.7 mL, decrease of 83% (decrease in analysis time of 57%)
2
3
45 6
Page 14
Flow rate column 2 = (diameter column 2)2/(diameter column 1)2 x Flow rate column 1
Quick Reference for Changing to Common Column Diameters
Maintains Equivalent Linear Velocity for Different C olumn ID’s:
Column Type Column ID Flow Rate
Analytical 4.6 mm 1.0 mL/min
Solvent Saver 3.0 mm 0.42 mL/min
NarrowBore 2.1 mm 0.21 mL/min
MicroBore 1.0 mm 47 µL/min
Capillary 0.5 mm 12 µL/min
Capillary 0.3 mm 4.2 µL/min
Nano 0.1 mm 472 nL/min
Nano 0.075 mm 266 nL/min
•Maintain equivalent mobile phase linear velocity when changing column diameter.
8
Page 15
Isocratic Tips and Tricks Summary
•Increased Isocratic Speed Does Not Necessarily Require Sub-Two Micron Particles
•Particle Diameter and Column Length are Major Factors in Resolving Power
• Chose New, Shorter Column to Approximate Original Column Efficiency - Chose Length Based on Time Savings Goal
Example 3X: 150mm, 5µm (~12,000N) ~ 50mm, 1.8µm (~12,000N)
•Usually Only Need to Reduce Col. Length and Particle Size –Not Necessary to Change Flow, Injection Vol. or Organic Content of M.P.
•Altered Column Diameter Requires Change in Flow and Injection Volume
Page 16
GRADIENT ELUTION
9
Page 17
Reducing Particle Size Improves KineticsReduces Peak Width, Increases Height- It Does NOT Ch ange the Gradient!
min2 4 6 8
mAU
0
200
400
600
800
1000
1200
1400
1600
Pressure at start: 51bar, 5µmRs(4,5) = 3.14Peak Width 5 σ (9) = 0.107
Pressure at start: 394bar, 1.8µmRs(4,5) = 5.07Peak Width 5 σ (9) = 0.074
�Chromatograms Offset for Better View
1.8um vs. 5um+ 60% Resolution (Theory: 67%)+ 160% Efficiency (Theory: 177%)
Page 18
VR ≈ N
4k*α
Resolution Equation for Gradient Elution Relationship of k* to
Gradient Time, Flow and Column Dimensions
tg FS (∆%B) Vm
k* ∝∆%B = difference between initial and final % B S = constant (≈ 4 for 100 - 500 Da)F = flow rate (mL/min.)tg = gradient time (min.)Vm = column void volume (mL)
Zorbax Vm= π x (Col Internal Radius)2 x Length x 0.6
10
Page 19
Expected: Use of a Longer Gradient Time Increases Gradient Retention
4
2
0 25 500 5 10 15Time (min)Time (min)
1,2
34 5
8
6,7
13
5
8
6
7
Column: ZORBAX SB-C84.6 x 150 mm, 5 µm
Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2
B: AcetonitrileFlow Rate: 1.0 mL/minTemperature:35°CSample: Herbicides
Gradient Time10 min.
Gradient Time60 min.
• Increased gradient retention improves resolution of several peak pairs – 1,2 and 4,5.
Page 20
Tip: A Shorter Column (smaller Vm) Also Increases Gradient Retention and Rs Increases Gradient Retention, Increases Overall Res olution
2
1,2
34 5
6
7
8
0 5 10 15Time (min)
0 5 10Time (min)
13
4
5
67
8
Column: ZORBAX SB-C8Gradient: 20 – 60% BMobile Phase: A:H2O with 0.1%TFA, pH 2
B: AcetonitrileFlow Rate: 1.0 mL/minTemperature:35°CSample: Herbicides
4.6 x 150 mm, 5 µm 4.6 x 75 mm, 3.5 µm
11
Page 21
300SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3 .5µm
Mobile Phase: A: 95% Water : 5 % ACN, 0.1% TFAB: 5% Water : 95% ACN, 0.085% TFAGradient: 10-60% B in 30 min.
Flow Rate: 1.0 mL / min.Temperature: Ambient
Sample: 1. Gly-Tyr 6. Leu-Enk2. Val-Tyr-Val 7. Angiotensin II3. [Gln 11] Amyloid- β- 8. Kinetensin
Protein Fragm 1-16 9. RNase4. (TYR8) Bradykinin 10. Insulin (Eq.)5. Met-Enk
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
1
2
3
45
6
7 8
9
10
12
3
45
6
7 8
9
10
000997P1.PPT
Tip: Improve Resolution By Using ShortColumn Length ( Vm ) for BioMolecules
Page 22
Vm-Column length
Vm-Column (i.d.)
Decrease in t G or F
Decrease in t G or F
Any Decrease in Can be Offset by a Proportional
k* ∝tG • F
S • ∆%B • Vm
001809S1.PPT
Tip: Maintain k* To Keep Relative Peak Position in a Chromatogram Unchanged While Reducing Time
12
Page 23
Time (min)
0 5 10 15 20 25
1
2
4
5
3
Time (min)
0 5 10 15
Column: StableBond SB-C84.6 x 150 mm, 5 µm
GradientTime: 30 min.
Flow Rate: 1.0 mL/min
AnalysisTime: 24 min
8
6
7
1
2
45
3
8
6
7
Column: Rapid Resolution StableBond SB-C84.6 x 75 mm, 3.5 µm
GradientTime: 15 min.
Flow Rate: 1.0 mL/min
AnalysisTime: 12 min
Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor
001784S1.PPT
Trick: Change V m and t G by Same Proportion
Two Chromatograms Both Having the Same Gradient Ste epness
Page 24
Very Fast LC on Conventional 1100 HPLC
G1379 Degasser
G1311 Quaternary pump
G1313A ALS autosampler
G1316A column compartment
G1314A VWD (standard cell G1314-60086, 10mm, 14uL)
AcetophenoneDiethyl phthalateBenzophenoneButyrophenoneValerophenoneHexanophenoneHeptanophenoneOctanophenone
The Sample
The Instrument
13
Page 25
0 2 4 6 8 10 12 14
mAU
0
50
100
150
200
250
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)
1.4
74
3.3
23
5.0
64
5.6
51
6.1
14
6.9
64
8.3
37
9.6
90
10.
982
Conventional Column - 4.6 x 150mm, 5µm, SB-C18
Flow Rate 1.0 ml/minInjection Volume 15uLTemperature 30°CWavelength 246nmSample rate 2.5 Hz
Time (min) % Acetonitrile
0 50
10 90
13.5 90
13.6 50
15 50
Initial Pressure: 69 barFinal Pressure: 38 bar
Page 26
Tip: Shorten Column and Gradient Time by Same Facto r1/3 Column Length- 1/3 Gradient TimeRRHT Column – 4.6 x 50mm , 1.8µm, SB-C18
min0 1 2 3 4 5 6
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
1.1
76
2.0
04
2.2
54
2.4
64
2.8
11
3.3
57
3.8
71
4.3
43
Flow Rate 1.0 ml/minInjection Volume 5uLTemperature 30°CWavelength 246nmSample rate 13.74 Hz
Time (min) % Acetonitrile
0 503.33 904.5 904.53 50
5 50
Initial Pressure: 132 barFinal Pressure: 74 bar
14
Page 27
The Comparison of chromatogram with the same RRHT column in 1100 and 1200 SL
min1 1.5 2 2.5 3 3.5 4 4.5 5
Norm.
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180008.D)
1.1
79
2.0
07
2.2
57
2.4
68
2.8
16
3.3
61
3.8
74
4.3
43
*DAD1 A, Sig=246,4 Ref=360,100 (WORKSHOP-TAIWAN\070815SBC180003.D)
Overlay the two Chromatograms
The red one is 1200, the blue one is 1100.
Page 28
Tip: Increase Column Flow-Reduce Gradient TimeDouble Flow (2mL/min) – ½ Gradient Time
RRHT 4.6 x 50mm, 1.8µm, SB-C18
0 0.5 1 1.5 2 2.5 3
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D)
0.5
87
1.0
20
1.1
46
1.2
60
1.4
37
1.7
15
1.9
70
2.2
01
Flow Rate 2.0 ml/minInjection Volume 5uLTemperature 30°CWavelength 246nmSample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
1.67 90
2.25 90
2.27 50
3.34 50
Initial Pressure: 266 barFinal Pressure: 146 bar
15
Page 29
Gradient Tips and Tricks Summary
Gradient Retention Relationship Formula Is the Key to -
• Improving Resolution
– Longer Gradient Time (tg)
– Smaller Column Volume (Vm) Length or Inner Diameter
– Keeping All other Parameters Constant
•Increasing Speed Without Losing Resolution
– Shorter Column with Proportionally Shorter Gradient Time
– Shorter Gradient Time with Proportionally Faster Flow Rate
tg FS (∆%B) Vm
k* ∝
Page 30
� Injection Volume Conversion
�Detector Settings recommendation
�Gradient and Isocratic Method Conversion (auto-detected)
Tip: Method Translator Makes Changes EasyTrick: Let Agilent Method Translator Do the MathBasic Mode for Easy Transfer of Conventional Method to RRLC
16
Page 31
Original Method New Method
Detailed OutputDetailed Input
Agilent Method Translator – Advanced ModeMore Detaled Information, But Still Easy to Use
Page 32
Analysis of impurities of an active pharmaceutical ingredient by conventional HPLC (4.6mm ID x 250 mm, 5.0 µm):
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40 OH
HN
CH3CH3
OCH3
Main Compound
OH
HN
CH3CH3
OCH3
Impurity A
O CH3
Br
Bromanisole
NCH3CH3
OCH3
Impurity B
CHCHN
33
OCH3
H
Impurity C
NCH3CH3
OH
H
OH
Impurity D
Does it work? - Example
17
Page 33
Converting to a 4.6 mm ID x 100 mm, 1.8µm column:
Page 34
Does it work? Yes!
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40 Conventional HPLC
min0 1 2 3 4 5 6 7 8
mAU
-2
0
2
4
6
8
10
12
14
Resolution optimized 1.8µm
Added Benefit of Increased Sensitivity!
18
Page 35
4.6 mmID x 250 mm, 5.0µm Zorbax SB C18
0.00 min 5% B20.00 min 90% B23.00 min 90% B23.01 min 5% B30.00 min 5% B
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
Conventional HPLC
4.6 mmID x 100 mm, 1.8µm Zorbax SB C18
0.00 min 5% B8.00 min 90% B9.20 min 90% B9.21 min 5% B
12.00 min 5% B
min0 1 2 3 4 5 6 7 8
mAU
0
5
10
15
20
25
30
35
Simple Conversion
min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
5
10
15
20
4.6 mmID x 100 mm, 1.8µm Zorbax SB C18
0.00 min 5% B4.33 min 90% B4.98 min 90% B4.99 min 5% B
6.5 min 5% B
Speed Optimized
Does it work?
Page 36
Eclipse PAH
ZORBAX Selectivity Choices Options for Improved Speed and Resolution
Extend-C18Extend-C18Bidentate-C18
Double endcapping
SB-C18Diisobutyl-C18
SB-PhenylDiisopropyl-
Phenyl
StableBondDiisopropyl
Diisobutyl bonding
Rx/SB-C8Diisopropyl-C8
SB-AQDiisopropyl
SB-C3Triisopropyl
Rx-C18Dimethyloctadecylsilane
Eclipse XDBEclipse XDBDimethyl bonding
Double endcapping
Eclipse XDB-C18
Eclipse XDB-C8
Eclipse XDB-Phenyl
Eclipse XDB-CN
Bonus-RPBonus-RPEmbedded amideDiisopropyl-C14Triple endcapped
Reversed Phase ChromatographyReversed-Phase Chromatography
SB-CNDiisopropyl-CN
Eclipse PlusEclipse PlusDimethyl bonding
Double endcapping
Eclipse Plus C18
Eclipse Plus C8
Eclipse Plus-Phenyl-Hexyl
Specialty products
19
Page 37
Tip: Bonded Phase Can Change SelectivityImprove Peak Shape and Shorten HPLC Assay Time
•Each ZORBAX phase available in matching 1.8µm,3.5µm and 5µm choices, most in 7µm!
•Over 140 RRHT,1.8um 600 bar column choices available
•14 Different Column Chemistries – 13 bonded phases and silica for HILIC use
•7 column lengths (250*, 150, 100, 75, 50, 30 and 20 mm long) * 1.8µm as custom column
•3 internal diameters (4.6, 3.0, 2.1 mm and Prep ID)
Page 38
Group/Presentation TitleAgilent Restricted
Month ##, 200X
Tip: C8 Often Yields Same Peak Order in < Time Than C18 Trick: Evaluate Different Phases with Same M.P.
1. Oxybenzone2. Internal Std.3. Octylmethoxycinnamate
Rapid Resolution Eclipse Plus C8, 4.6 x 50mm, 3.5 u m
Conditions:Mobile Phase.: Water: Acetonitrile (30:70)Flow Rate: 2.0 mL/min. Detection: UV 230nm Temperature: 30 °CSample: Lip balm extract in ACN(melted at 100°C ACN , cooled and 0.45 um filtered)
2.2 min.
RRHT Eclipse Plus C18, 4.6 x 50mm, 1.8 um
N: 6600Pw: 0.037 min.TF: 0.99
N: 6300Pw: 0.061 min.TF: 0.98
N: 5000Pw: 0.019 min.TF: 1.09
N: 8800Pw: 0.014 min.TF: 1.20
N: 11600Pw: 0.047 min.TF: 1.06
N: 11800Pw: 0.093 min.TF: 1.02
247 bar
88 bar
min0 1 2 3 4
min0 1 2 3 4
)
Less retention can save significant time – the C8 is a good choice here.The RRHT column is delivering the efficiency and re solution expected, but the C8 bonded phase may be the best choice.
20
Page 39
Tip: Polar Phases Can Save Analysis TimeTrick: Evaluate Non-Polar and Polar Phases with Sam e M.P.
Group/Presentation TitleAgilent Restricted
Month ##, 200X
Eclipse Plus Phenyl Hexyl
Eclipse Plus C8
Eclipse Plus C18
Mobile Phase 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH 2.4 Flow Rate: 1.5 ml/min 4.6 x 50mm UV 210 nm 2µl Elution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5) Ibuprofen, (6) Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions
min0 2 4 6 8 10
min0 2 4 6 8 10
min0 2 4 6 8 10
Elution order reversal between Phenyl-Hexyl and Alk yl chains
Page 40
Tip: Higher Temperature Can Improve Speed and Resolution
Higher Temperature should always be considered
as a parameter during speed optimization
•Provides more rapid mass transfer:– Improves Efficiency – enhances resolution
– Decreases analysis time – faster separations with no loss in resolution
•Decreases Mobile Phase Viscosity– Lowers backpressure – allows for higher flow rates, faster separations,
greater efficiency
•Can change selectivity – optimize resolution
•Not Necessary to Go to the Limit-Midrange Offers Benefits
21
Page 41
Tip: Temperature Changes Can Alter SelectivityTrick: Optimize Col Temp For Best Speed/Rs
20°C
60°C
40°C
30°C
90°C
Salicylic acid
min0 1 2
0 1 2 345
0 1 2 345
0 1 2 345
0 1 2345
3 4
Salicylic acid
Salicylic acid
Salicylic acid
Salicylic acid
Column: RRHT SB-C184.6 x 50mm, 1.8um
Coelution
Increased T Decreases Run Time, Changes Selectivity and Sharpens Peaks
Improving Resolution
Page 42
Tips on When You Should Move to the Higher Pressure and Performance 1200 SL (RRLC) System
•Very Fast Methods with High Velocity Mobile Phase That Increase Backpressure Above 400bar
•Resolution of Difficult Samples Requires Long Columns With 1.8µm Particles (>400bar Backpressure)
•Labs Required to Run Both Traditional and New, Higher Performance LC Methods
•Need Faster Data Acquisition for Narrow, High Efficiency Peaks Generated in Higher Speed Methods
22
Page 43
Tip: 1.8µm Particle Increases Resolution (How?)Trick: Substitute 1.8µm Particles in Same Length Co lumn
Ref: Appl. Note 5989-4506 by Edgar Naegele
Conditions for both experimentsPumps• Solvent A: H2O + 0.1% TFA Solvent B: ACN + 0.1% TFA
• Gradient: 10% to 95% ACN in 40min, hold for 1min
• Flow Rate: 0.4ml/minAutosamplers• Injection volume: 3µlThermostatted Column Comp.• Temperature: 50°CDetectors• DAD 2µl cell and 20Hz, 220nm,
Agilent 1200 RRLC
min13 14 15 16 17 18
50
100
150
200
250
15.32
415.16
4 16.74
615.47
215.79
0mAU
15.07
914.93
714.78
7
Zorbax 2.1x150mm SB C-18, 1.8µm
min13 14 15 16 17 18
mAU
50
100
150
200
250
300
350
17.13
6
15.41
1
15.96
7
15.15
9
15.55
5
15.06
5Agilent 1100 Zorbax 2.1x150mm SB C-18, 5µm
Page 44
min0 5 10 15 20
mAU
-1
-0.5
0
0.5
1
1.5
2
2.5
DAD1 A, Sig=254,4 Ref =of f (051119A\SIG10003.D) Column:150x4.6 mm
5µm
Pressure: 93 bar
N: 8213
Height: 1.25 mAU
S/N: 42.3
Rs = 1.15
tr = 14.9 min (1st epimer)
Nptp: 2.4 � 10-2 mAU
4.6 x 150, 5um
93 bar
N = 7259
R_S = 1.15S/N = 42
Height = 1.25
Noise = 24uAU
Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 � 10-2
min10
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
3
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 � 10-2
min10
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
3
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 � 10-2 mAU
min0 5 10 15 20
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 � 10-2 mAU
min0 5 10 15 20
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 � 10-2
min10
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
3
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Column:150 x 4.6 mm 1.8µm
Pressure: 490 bar
N: 28669
Height:1.78
S/N: 43.6
Rs = 1.80
tr = 17.2 (1st epimer)
Nptp: 3 � 10-2
min10
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
3
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 � 10-2 mAU
min0 5 10 15 20
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
Column:150x4.6 mm
3.5µm
Pressure: 165 bar
N: 14862
Height:1,34 mAU
S/N: 50.7
Rs = 1.37
tr = 15.3 min (1st epimer)
Nptp: 2 � 10-2 mAU
min0 5 10 15 20
Norm.
-1
-0.5
0
0.5
1
1.5
2
2.5
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
4.6 x 150, 3.5um
165 bar
N = 14862
R_S = 1.37S/N = 50
Height = 1.34
Noise = 20uAU
4.6 x 150, 1.8um
490 bar
N = 28669
R_S = 1.80 (+57%)S/N = 44
Height = 1.80
Noise = 30uAU
1/dp
0.2 0.3 0.4 0.5 0.6
N
5000
10000
15000
20000
25000
30000
pdN
1∝
Tip: Small Particles Improve Detection of Low Level Impurities
23
Page 45
Particles Reveal More Information and Improve Detection and Integration
4.6 x 150, 1.8µm490 barN = 28669 R_S = 1.80 (+57%)S/N = 44
7 Impurities
All 7 Baseline Separated!
4.6 x 150, 3.5µm165 barN = 14862R_S = 1.37S/N = 50
7 Impurities
6 Not Baseline Separated!
4.6 x 150, 5µm93 barN = 7259R_S = 1.15S/N = 42
4 Impurities
2 Not Baseline Separated!
Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7min)
Up to 60% higher resolutionwithout loss in sensitivity
Page 46
F= 1.20ml/minT = 40°CAnalysis Time = 11minSolvent Cons. = 13.2ml
High Resolution:4.6mm x 150 mm 5.0µm
min0 2 4 6 8 10 12
F = 4.80ml/minT = 40°CAnalysis Time = 1.05minSolvent Cons. = 5.1ml
High Speed:4.6mm x 50 mm 5.0µm
min0 0.2 0.4 0.6 0.8 1
F= 1.00ml/minT = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml
High Speed & Resolution:2.1mm x 50mm 1.8µm
min0.2 0.4 0.6 0.8 10
Example of What Is Possible!Shorter Column, Smaller Particle, > Flow Rate, High Temp
Max Speed at T = 95 oC2.1mm x 50mm 1.8um
F= 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml
PWHH = 197msec
min0.2 0.4 0.6 0.8 10
> 20x faster !
Easy!
Easy!
Easy, But You Will Need > 400bar!
Easy!
24
Page 47
Data Rate
Peak Width
Resolution Peak Capacity
80 Hz 0.300 2.25 60
40 Hz 0.329 2.05 55
20 Hz 0.416 1.71 45
10 Hz 0.666 1.17 29
5 Hz 1.236 0.67 16
min0.1 0.2 0.3 0.4 0.50
80Hz
PW=0.30sec
40Hz
PW=0.33sec
20HzPW=0.42sec
10HzPW=0.67sec
5HzPW=1.24secSample: Phenones Test MixColumn: Zorbax SB-C18, 4.6x30, 1.8umGradient:: 50-100%ACN in 0.3minFlow Rate: 5ml/min
80Hz versus 10Hz Data Rate• Peak Width: – 55%• Resolution: + 90%• Peak Capacity: + 120%• App. Column Eff.: + 260%
Tip: Higher Data Improves Response and Rs Trick: Optimize Data Rate for Accurate Profile
Page 48
Moving From Conventional LC to RRLC:
Moving to RRLC is simple and easy !Moving to RRLC is simple and easy !
• Same software (e.g. ChemStation, EZChrom)
� No additional training effort
• Limit capital investment by modular step-wise upgra de
� Leverage existing 1100 modules, existing method
and stationary phase
• Configurable delay volume in 1200 SL pump
� Easy to also run legacy LC methods
• Simple LC to RRLC method translator
� No tedious method development
“Doesn’t this cost a fortune ?”
“No - not at all !”
25
Page 49
Stepwise Scale-up to Rapid Resolution LCFrom 1100 to 1200 RRLC in two steps -Add What You Need When You Need It!
Actual: 1100
Quat System
> 5 min
> 6 min> 3 sec
5 - 12,0004.6 mm
50 mm 0.2 - 10ml/min
80 C400bar
ALS
TCC
VWD/MWD/DAD
Quat Pump
Degasser
Step 1: 1100/1200
„Bin SL“ System
h-ALS SL
TCC
VWD/MWD/DAD
Bin Pump SL
µ-Degasser
> 1.5min
> 2min> 1.5 sec
5 - 30,0002.1 - 4.6 mm
50 - 150 mm0.05 - 5 ml/min
80 C600 bar
Analysis Time
Cycle timesPeak Width
NColumn ID
Column Length Flow rates
TemperaturePressure
Step 2: 1200
Rapid Resolution System
h-ALS SL
TCC SL
DAD SL
Bin Pump SL
u-Degasser
> 0.2min
> 0.4min> 0.2 sec
5 - 60,0002.1 - 4.6 mm
20 - 150mm0.05 – 5 ml/min
100 C600bar
+ Speed+ Resolution+ Sensitivity
+ MS-Robustness+ Data Security & Traceability
+ Qualification (Degasser, ACE)+ Compatible with conv. HPLC
+ Resolution+ Speed
+ MS-Compatibility+ Solvent Saving
+ Compatible with conv. HPLC
Page 50
SUMMARY
• Increasing Speed of HPLC Separations Need Not be Difficult or Costly
•Changes in Columns and Equipment Depend on Your Goals
•Majority of Time Saved Will Be Based on Column and Particle Size
•Isocratic and Gradient 2X , 3X and 5X Times Savings are Easy
•Greater Time Savings May Require Instrument Modifications
•10-20X Increase in Speed May Require Instrument Upgrade
•Long Columns with 1.8µm Particles May Require >400 bar Pump and Autosampler Capability